Costimulatory receptors and their ligands have a profound impact on autoimmune diseases by regulating the balance between pathogenic (Th1 and Th17) and CD4+.CD25+, Fox-P3+ T-reg cells. We recently proposed that there is a reciprocal relationship between Treg and Th17 cells phenotypes and their transcriptional factors that control the generation of pathogenic and regulatory T cells. TFGD supports FoxP3+CD4+ T cells, but IL-6 inhibits the generation of T-reg cells and induces Th17 cells instead. IL-23 produced by activated antigen presenting cells further strengthens the reciprocal dichotomy in terms of suppressing the Fox-P3 induction and enhancing Th17 response. Therefore, APCs present in the lymphoid organs or in target tissues play an important role in determining the balance between T cell phenotypes, via their production of cytokines and the costimulatory molecules that they express. We hypothesize that costimulatory molecules and the receptors influence the generation of pathogenic and regulatory T cells and thus regulate development of autoimmune disease. We propose these specific aims to address this hypothesis: 1.Study the role of costimulatory receptors (CTLA4, ICOS or PD1) in the induction and differentiation of regulatory (CD4+, FoxP3+ Treg) and effector (Th1 and Th17 cells) in vitro and in the induction and regulation of EAE and diabetes in vivo. Experiments in this aim will combine transgenic TCRs, tetramer reagents to specifically track T cells with an autoimmune specificity, fluorescent reporters mice that will allow us to follow the in vivo phenotype of differentiating effector and regulatory T cells. 2. In parallel experiments to aim 1, we will study the impact of costimulatory molecules expressed on the APCs in the differentiation of regulatory and pathogenic effector T cells in vitro and in vivo. The effect of loss or blockade of the costimulatory molecules on APCs function will be tested by analyzing the changes in the expression of cell surface molecules and cytokines that the APCs and stromal cells produce and thus influence T cell differentiation and development of EAE and diabetes. 3. Assess in depth the phenotypic variation by genomic profiling in aggressive and regulatory populations present in the target organ during development of EAE and diabetes. Our preliminary data suggests that progression of disease is not simply due to lower frequencies of Treg cells, but that both effector and regulatory T cells in the target tissue change in response to inflammatory and costimulatory cues. Using the expression profiling we will determine the changes in the T-reg and T-effector populations present in the target tissue and how these cells change during costimulatory blockade. The experiments presented here, will address the role of costimulatory molecules and their receptors in regulating the balance of pathogenic and regulatory T cells in the lymphoid tissue and the target organ.